The overall goal of this project is to elucidate the molecular mechanisms by which c-FLIP mediates the apoptotic and anti-apoptotic activities that are critical for TRAIL function and to determine the basis for tumor selectivity of TRAIL-induced apoptosis in vivo. Our experimental plan is designed to test our central hypothesis that c-FLIP is a multifunctional protein acting as a critical molecular on/off switch regulating TRAIL-induced apoptosis. The Specific Aims of this proposal are: 1) determine the role of c-FLIP in TRAIL-induced apoptosis; 2) determine the signal transduction pathways that mediate c-FLIP function; and 3) analyze role of c-FLIP in tumor selectivity of TRAIL-induced apoptosis and to analyze tumor-selective apoptosis by TRAIL and TRAIL-induced pathways in vivo. The biological role of c-FLIP in mediating TRAIL function is mostly unknown. In Aim 1, we will determine the mechanistic function of c-FLIP in TRAIL-induced apoptosis in vitro using cells stably expressing c-FLIP and in vivo using B and T cells of c-FLIP mice. These mice are generated by transplantation of bone marrow that is retrovirally infected with c-FLIP in lethally irradiated recipient mice. Finally, we will carry out structure and function analysis of c-FLIP to determine the role of DED domains and the inactive caspase domain of c-FLIP in mediating its pro- versus anti-apoptotic function. We have found that the anti-apoptotic pathways NIP-KB, Akt and pro-apoptotic protein caspase-8 are activated in response to c-FLIP. In Aim 2 of this proposal, we will determine the requirement of these proteins in mediating c-FLIP and TRAIL function. This will be done using dominant active and dominant inhibitory approach in cell culture studies and in bone marrow transplanted mice. In addition, we will utilize the Jurkat cells that are deficient in caspase-8 to define the role of caspase-8 in mediating c-FLIP function. TRAIL selectively induces apoptosis in transformed/tumor but not in normal cells in vitro, however the tumor selectivity of TRAIL-induced apoptosis in vivo is not known. In Aim 3, we will analyze the role of c-FLIP in selective tumor death by TRAIL. We will also determine the ability of TRAIL and c-FLIP to selectively and specifically regress the Bcr/Abl-induced CML without killing the normal cells in mice that are generated by bone marrow transplantation. We will analyze the signs of CML by histological analysis of spleen and bone marrow specimens, and will determine the tumor selectivity of apoptosis by measuring it in various tissues.